KR20210120034A - Antisense drug against human intracellular adhesion molecule 1 (ICAM-1) - Google Patents

Abstract

The present invention relates to specific antisense oligonucleotides that inhibit the expression of the adhesion molecule ICAM-1 in human cells. The present invention also relates to vectors containing said oligonucleotides and host cells containing said vectors or oligonucleotides and pharmaceutical compositions containing said oligonucleotides, as well as their use, particularly in the treatment of inflammatory diseases or conditions in humans. .

Description

Antisense drug against human intracellular adhesion molecule 1 (ICAM-1)

The present invention relates to specific antisense oligonucleotides that inhibit the expression of the human intercellular adhesion molecule ICAM-1. The present invention also relates to vectors containing said oligonucleotides and host cells containing said vectors or oligonucleotides and pharmaceutical compositions containing said oligonucleotides, as well as their use, particularly in the treatment of inflammatory diseases or conditions in humans. will be.

Antisense oligonucleotides (asONs) are synthetic single-stranded nucleic acids typically ranging in length from 16 to 24 nucleotides. It binds to single-stranded target messenger RNA (mRNA) through Watson-Crick interaction in a sequence-specific fashion, thus inhibiting target gene expression mainly at the post-transcriptional level, thereby interfering with gene expression. Indicates the main tools.

Since the first report on asON in 1978 (see Zamecnik, PC, Stephenson, ML, Proc. Natl. Acad. Sci., US A., Vol. 75, pp. 280-284 (1978)), (i) extended half-life in the target cell and target environment, (ii) biological efficacy by increasing the stability of complexes with target mRNA, (iii) improved delivery to target cells and tissues, and (iv) reduced e.g., reduced A wide variety of chemical modifications have been developed that improve efficacy by a number of properties, including non-specific toxicity and undesirable side effects such as immune-stimulation. As such, all moieties of oligonucleotides (ie, sugar moieties, nucleobases, and internucleotide phosphate backbones) have been subjected to chemical modifications.

Over the past few decades, three to four so-called "chemical generations" for asON have been developed (Deleavey, GF, Damha, MJ, Chemistry & Biology, Vol. 19, pp. 937-954 (2012); Khvorova). , A., Watts, JK, Nat. Biotechnol., Vol. 35, pp. 238-248 (2017)). In general, more advanced generations of asON are characterized by incremental improvements in the above-mentioned representative features of asON. Comparison of the extent of antisense effect by a related set of chemically modified asONs is LNA (locked nucleic acid) > phosphorothioate-modified backbone > 2'-OMe-modified sugar (see Figure 1 and Table 1 as well as the literature: Grunweller, A. et al., Nucl. Acids Res., Vol. 31, pp. 3185-3193 (2003)), clearly shows the order of efficacy.

The second important prerequisite for the high efficacy of asON is related to the local structure of the target RNA and its accessibility (Bo, X. et al., BMC Bioinformatics 7:122, (2006); Schubert, S. et al., J. Mol. Biol. Vol. 348, pp. 883-893 (2005); Sczakiel, G., Kretschmer-Kazemi Far, R., Curr. Opin. Mol. Ther., Vol. 4 , pp. 149-153 (2002)). These factors cannot be changed once the local target site is defined. Thus, for a given good local target site, efficacy can only be improved by the properties of the selected antisense molecule, ie by modifying its chemistry.

Typically, the biological efficacy of asON is tested in an appropriate cellular system. In the case of human target genes, testing begins with the appropriate human cell line. In general, efficacy is compared using the dose of inhibitor that results in 50% target inhibition (IC _{50 value).} However, in the case of oligonucleotide-based drugs/inhibitors, the dose-response relationship may be irregular. This means that sometimes 100% of target gene inhibition cannot be achieved or that the inhibition is not dose dependent. Therefore, we characterize the biological effect of asON by preferably using more parameters. Thus, inhibition studies of the use of asON against a given target are often characterized by (i) "maximum achievable degree of target inhibition", (ii) a concentration of one-half the maximal target inhibition (IC ₅₀ value), (iii) a given It is characterized by the extent of target inhibition at the asON concentration (eg, 100 nM), and (iv) the overall dose-response relationship. These important parameters of target inhibition often change in an irrational fashion. For this reason, a generic description of the action of antisense oligonucleotides and control oligonucleotides is generally considered.

A target of particular interest for asON is the mRNA of the gene for human intracellular adhesion molecule 1 (ICAM-1; CD54), eg to prevent or treat inflammatory processes in humans. ICAM-1 already exhibits basal gene expression in healthy cells, often about 40-50% of the stimulated expression in inflammation. When inhibiting ICAM-1 to prevent or inhibit inflammation in humans, this may lead to undesirable side effects, so it may be desirable not to fall below a healthy baseline expression level. ICAM-1 inhibitor antisense nucleic acids may not cause side effects due to toxic or non-specific immunostimulatory effects and may be the most cost-effective to produce.

A large diversity of asONs to ICM-1 has been described so far (see, e.g., Chiang, MY. et al., J. Biol. Chem., Vol. 266, pp. 18162-18171 (1991); Patzel, V. et al., Nucl. Acids Res., Vol. 27, pp. 4328-4334 (1999)). However, these asONs have different drawbacks, such as non-optimized inhibitory effects (eg poor potency or inhibition under basal expression levels), toxicity, non-specific immunostimulatory effects (eg CpG motifs). ), and large production costs.

Therefore, the technical problem underlying the present invention is a means for suppressing the gene expression of ICAM-1 close to the basal expression level, and a means that has low toxicity and non-specific immunostimulatory effect on human cells and is accessible at low production cost is to provide

The solution to the above technical problem is achieved by the embodiments characterized in the claims.

In particular, in a first aspect, the present invention relates to an antisense oligonucleotide comprising the nucleotide sequence according to SEQ ID NO: 1 or a fragment thereof. The antisense oligonucleotide of the present invention particularly relates to a specific mRNA sequence of ICAM-1 as a target molecule, and is preferably capable of binding to positions 1841 to 1860 of ICAM-1 mRNA (HSICAM01:J03132).

In this regard, the specific antisense oligonucleotides used herein can advantageously inhibit stimulated ICAM-1 expression and at the same time preferably (significantly) inhibit the level of basal ICAM-1 expression in humans. none. In particular, stimulated ICAM-1 expression to a greater extent as in the previously known asON targeting ICAM-1, which under experimental conditions has no efficacy leaving more than 30% to 35% of gene expression in the IL-1β-stimulated range. It is possible to suppress Using the antisense oligonucleotides of the present invention, preferably the IL-1β-stimulated range of ICAM-1 gene expression is not already known in general for major improvements in potency or even known for lower potency. , it is possible to down-regulate up to 40% and more than -10% even in the specific case of chemical modification of the first/second generation.

In a preferred embodiment, the antisense oligonucleotides of the present invention are for use in a method of preventing or treating a disease or condition in a human, wherein the disease or condition is associated with overexpression of ICAM-1. Preferably, the disease or condition is an acute or chronic inflammatory disease or condition, viral infection, metastasis, inflammation of the skin, mobilization of hematopoietic stem cells, coryza, ulcerative colitis, rheumatoid arthritis ( rheumatoid arthritis), lupus erythematosus, organ rejection reaction, graft-versus-host reaction to allogeneic bone marrow transplantation, psoriasis, asthma, and neurodermatitis (neurodermatitis). Preferably, the disease or condition is an inflammatory disease or condition of the oral cavity in a human. Preferably, the inflammatory disease or condition of the oral cavity to be prevented or treated is chronic inflammation of the gums, such as periodontal disease or condition, more preferably gingivitis, chronic periodontitis, aggressive periodontitis, periodontitis and peri-implantitis. Implantitis) or prevalence of systemic diseases such as mukosivitis, necrotizing ulcerative gingivitis, necrotizing ulcerative periodontitis, abscesses of the periodontium, and complex periodontology A periodontal disease or condition selected from the group consisting of a combined periodontic-endodontic lesion.

As used herein, the term "oligonucleotide" refers to natural, semi-synthetic, synthetic or (chemically) modified, single or double of deoxyribonucleotides and/or ribonucleotides and/or modified nucleotides. It relates to a stranded nucleic acid molecule. Suitable modified nucleotides include nucleotides with modified base, sugar, and/or phosphate groups. Methods for preparing, isolating and/or purifying oligonucleotides are known in the art and include chemical synthesis or enzymatic methods of oligonucleotides and subsequent purification. In a preferred embodiment, the oligonucleotide is a single- or double-stranded oligonucleotide, preferably a single-stranded modified oligonucleotide of the chemistry outlined above, more preferably a gapmer oligonucleotide.

The term "gapmer oligonucleotide" as used herein refers to a central single-stranded oligonucleotide flanked at one or both ends by single-stranded segments of different chemical composition, for example, by a 2'-O-methyl RNA oligonucleotide sequence. Refers to an oligonucleotide comprising a. Each of these terminal oligonucleotide sequences preferably has a length of 2 to 7, more preferably 4 oligonucleotides. Preferably, the gapmer oligonucleotide is a second generation RNA/DNA gapmer oligonucleotide comprising a 2'-0-methyl recognition arm and a nucleic acid core, thereby triggering specific RNAse H cleavage.

As used herein, the term "fragment of a nucleotide sequence according to SEQ ID NO: 1" refers to a biological sequence of a nucleotide sequence according to SEQ ID NO: 1 having at least 10, at least 12, at least 15, or 18 nucleotides of SEQ ID NO: 1 means an active fragment, ie, the fragment has 10, 12, 15, or 18 nucleotides of SEQ ID NO:1. In a specific embodiment, the oligonucleotide consists of the fragment, ie, the oligonucleotide has a length of 10, 12, 15, or 18 nucleotides. The oligonucleotide comprising the fragment is preferably at least 10, 12, 15, or 18 nucleotides, and at least 50 nucleotides, more preferably 40 nucleotides or less, more preferably 35 nucleotides or less, more preferably no more than 30 nucleotides, and most preferably no more than 25 nucleotides. The term “biologically active” refers to such fragments that bind to a specific RNA sequence of ICAM-1 as a target molecule and inhibit stimulated gene expression and preferably do not (significantly) inhibit basal gene expression of ICAM-1. refers to the ability of

In a particularly preferred embodiment, the oligonucleotide of the invention comprises the entire nucleotide sequence according to SEQ ID NO:1. Preferably, said oligonucleotide consists of said nucleotide sequence, ie said oligonucleotide has a length of 20 nucleotides. The oligonucleotide comprising the nucleotide sequence is preferably 20 to 50 nucleotides, more preferably 20 to 40 nucleotides, more preferably 20 to 35 nucleotides, more preferably 20 to 30 nucleotides, and most Preferably it has a length of 20 to 25 nucleotides.

As described above, the antisense oligonucleotides of the present invention are capable of down-regulating a stimulated range, preferably an IL-1β-stimulated range, of ICAM-1 gene expression. The stimulated range of ICAM-1 gene expression, preferably the IL-1β-stimulated range, is preferably 100% stimulated, preferably IL-1β-stimulated, based on ICAM-1 gene expression, 40% or less, more preferably 30% or less, more preferably 20% or less, more preferably 10% or less, more preferably 8.0% or less, more preferably 6.0% or less, more preferably 5.0% or less, and most preferably down-regulated to 4.0% or less. Moreover, the antisense oligonucleotides of the present invention are preferably unable to simultaneously (significantly) inhibit the level of basal ICAM-1 expression in humans. Thus, the stimulated range, preferably the IL-1β-stimulated range, of ICAM-1 gene expression is preferably 100% stimulated, preferably IL-1β-stimulated, based on ICAM-1 gene expression, It is down-regulated to -10% or less, more preferably -5.0% or more, more preferably -3.0% or more, more preferably -1.0% or more, and most preferably 0.0% or more.

In another aspect, the invention relates to a vector comprising an antisense oligonucleotide according to the invention. The above descriptions and definitions apply equally to this aspect of the invention. The term “vector,” as used herein, relates to any vector for the transport of a nucleic acid into a cell. In particular, the term includes plasmid vectors, viral vectors, cosmid vectors, and artificial chromosomes, wherein plasmid vectors are particularly preferred. Representative vectors are known in the art. Moreover, the term "vector comprising an antisense oligonucleotide according to the invention" includes a vector comprising a sequence complementary to an antisense oligonucleotide according to the invention, which after transcription produces an antisense oligonucleotide according to the invention .

In a further aspect, the present invention relates to a host cell comprising a vector and/or an antisense oligonucleotide of the present invention. The above descriptions and definitions apply equally to this aspect of the invention. Representative host cells are known in the art. These include, for example, suitable bacterial cells, yeast cells, plant cells, insect cells, and mammalian cells.

In another aspect, the invention relates to a pharmaceutical composition comprising a pharmaceutically active amount of an antisense oligonucleotide according to the invention, and optionally a pharmaceutically acceptable carrier, excipient or diluent. The above descriptions and definitions apply equally to this aspect of the invention.

The term "medicament" as used herein relates to any pharmaceutical composition comprising at least an antisense oligonucleotide according to the present invention in a pharmaceutically active amount.

In accordance with the present invention, the pharmaceutical composition may be administered by any route of administration known in the art to be suitable for delivering a medicament to a human. The route of administration does not represent a particular limitation and includes, for example, oral application, dermal application, topical application, other forms of topical application (eg, use of a delivery device). The pharmaceutical composition of the present invention is preferably capable of releasing oligonucleotides in tissues, in particular, for example, mucosal tissues.

The concentration of the antisense oligonucleotide in the pharmaceutical composition of the present invention is not particularly limited. For example, the concentration of antisense oligonucleotide in the pharmaceutical composition is 1.0 pM to 10 M, preferably 0.10 μM to 5.0 M, more preferably 1.0 μM to 500 mM, more preferably 10 μM to 500 μM, even More preferably, it may be 50 μM to 200 μM.

In a further aspect, the invention relates to a kit comprising an antisense oligonucleotide according to the invention. The foregoing descriptions and definitions apply equally to this aspect of the invention. In a preferred embodiment, the kits of the invention further comprise suitable buffers and/or suitable dispersible and/or suitable enzymes.

In a further aspect, the invention relates to the use of an antisense oligonucleotide according to the invention for reducing the expression of ICAM-1. The above descriptions and definitions apply equally to aspects of the present invention. In a preferred embodiment, it is used in vitro.

In a further aspect, the present invention relates to a method for reducing the expression of ICAM-1 comprising the step of introducing an antisense oligonucleotide according to the present invention or a vector according to the present invention into a human cell. The above descriptions and definitions apply equally to this aspect of the invention. In a preferred embodiment, the method is performed in vitro.

The present invention relates to the following nucleotide sequences.

N = 2'-deoxy nucleoside;

mN = 2'-0-methyl nucleoside;

5mC = 2'-deoxy-5-methyl-cytidine;

m(5mC) = 2'-0-methyl 5-methyl-cytidine;

All intranucleotide phosphates are monophosphorothioate-modified.

In the following SEQ ID NOs: 2 to 12, the following abbreviations apply:

L = LNA-modified nucleotides (LNA, locked nucleic acid);

F = 2'-fluoro-modified ribose;

M = 2'-0-methyl-modified ribose;

* = phosphate in phosphorothioate-modified nucleotides;

The drawing shows:
1 : Sequence of performance of modified asON derived from Grunweller et al. (Grunweller, A. et al., Nucl. Acids Res., Vol. 31, pp. 3185-3193 (2003)) : LNA > phosphorothioate > O-methyl gapmer.In this set, siRNA is the most potent inhibitor.This order is indicated by the dose-response relationship.The abbreviation for asON modified from the original literature is here for clarity. is fully described in
The invention will be further illustrated in the following non-limiting examples.

Substances and methods

oligonucleotide

All oligonucleotides were obtained from commercial suppliers. Quality control includes UV absorption measurement and integrity check by gel electrophoresis. The yield and purity of all oligonucleotides were measured by UV absorption spectroscopy and the integrity was controlled by staining with 20% denatured polyacrylamide gel followed by Stains-All (SIGMA-ALDRICH, Daisenhofen, Germany).

Cell Lines and Cell Cultures

The effect of oligonucleotides on the expression of the endogenous target gene intercellular adhesion molecule 1 (ICAM-1; CD54, Cluster of Differentiation 54) was measured using the human cell line ECV304. DNA fingerprinting according to the DSMZ-German Collection of Microorganisms and Cell Cultures (Brunschweig, Germany) showed that ECV304 expresses ICAM-1 in an inducible manner in the human urinary bladder. It is a derivative of the carcinoma cell line T-24 (Dirks, WG et al. In Vitro Cell Dev. Biol., Vol. 35, pp. 558-559, (1999)).

ECV304 cells were buffered with 25 mM Hepes pH 7.4 and medium 199 (SIGMA-ALDRICH, Daisenhofen, Germany) supplemented with 0.68 mM L-glutamine and 10% FCS (fetal bovine serum, Invitrogen, Karlsruhe, Germany). material) was kept. Cells were routinely split 2 to 3 times 1 week after trypsinization. For stimulation of ICAM-1 (CD54) 200 U/ml interleukin 1β (IL-1β; PromoCell, Heidelberg, Germany) was added to the medium and cells were incubated overnight (16-18 hours).

Inhibitory properties of antisense oligonucleotides in the IL-1β-induced state of ICAM-1 gene expression in human ECV-304 cells

Transfection of cells using asON

15 hours before oligonucleotide treatment, ECV304 cells were seeded in 12-well culture plates at ^{a density of 1.5} ×10 5 cells/well. Cells were then washed once with pre-warmed (37° C.) serum-reduced Opti-MEM I medium (Invitrogen, Kalluge, Germany). To deliver asON to cells, two transfection protocols were used: (1) transfection with cationic lipid DOTMA/DOPE (Lipofectin; Invitrogen/Thermo Fisher, Rockford, USA) per well. 1 ml of Opti-MEM I medium containing 0.1 μM asON or siRNA and 5 μg/ml of lipofectin was used. (2) Using Lipofectamine 2000 (Invitrogen/Thermo Fisher, Rockford, USA), transfection of asON was performed with 0.4 ml of Opti- containing 0.1 μM asON and 10 μg/ml of Lipofectamine 2000 per well per well. This was done using MEM I medium. Cells were incubated for 4 hours at 37° C., 5% CO _{2 .} Subsequently, the transfection medium was replaced with medium 199 containing 10% FCS. After incubation at 37° C., 5% CO ₂ for 4 hours, the medium was replaced again with medium 199 containing 10% FCS and 200 U/ml IL-1β to stimulate ICAM-1 expression. For immunofluorescence assays, after stimulation with IL-1β, cells were trypsinized from culture dishes with 100 μl of 0.25% trypsin/0.02% EDTA in PBS (phosphate buffered saline) for 5 min at 37°C for 16-18 h. was removed from the culture. ICAM-1-specific gene expression in cells treated with IL-1β and control asON or siRNA was set at 100%, whereas gene expression in cells treated with control asON or siRNA alone was defined as the basal expression level.

Quantification of ICAM-1-specific target proteins: immunofluorescence staining and flow cytometry

Cells were washed with phosphate-buffered saline (PBS, pH 7.4) and phycoerythrin (PE)-conjugated CD54 monoclonal antibody (clone LB-2) or homozygous PE control (both, Becton Dickinson, Germany) Heidelberg) in 50 μl of PBS containing 1% BSA (bovine serum albumin) at 4° C. for 30 minutes. Subsequently, the cells were washed with PBS and suspended in PBS containing 1% paraformaldehyde, pH 7.4. Cells were analyzed using Becton Dickinson FACSCalibur and CellQuest Pro software (Becton Dickinson, Heidelberg, Germany). After gating out dead cells, mean (PE)-fluorescence intensity was measured and inhibition of ICAM-1 gene expression was normalized to the IL-1β-induced state set to 100%. The basal gene expression level was approximately 35%.

Quantification of ICAM-1-specific target RNA: quantitative RT-PCR

Total RNA was extracted from the transfected cells using the RNeasy mini kit including treatment with RNase-free DNase I (Qiagen, Hilden, Germany). The yield and purity of RNA were determined spectrophotometrically. Synthesis of cDNA was performed according to the manufacturer's specifications (Invitrogen/Thermo Fisher, Rockford, USA) using random hexamer primers and Superscript II RNase H-reverse transcriptase. Quantitative PCR was performed using a GeneAmp 5700 Sequence Detection System (Applied Biosystems/Thermo Fisher, Rockford, USA) and SYBR Green PCR Core Reagent (Eurogentec, Seraiing, Belgium). ICAM-1 cDNA was amplified with a forward primer 5'-GCCACTTCTTCTGTAAGTCTGTGGG-3' and a reverse primer 5'-CTACCGGCCCTGGGACG-3' to generate a 300 bp fragment. A product of 258 bp was obtained by normalizing the sample with primers specific for cDNA encoding human GAPDH (forward primer, 5'-AACACGCGACACCCACTCCTC-3' and reverse primer, 5'-GGAGGGGAGATTCAGTGTGGT-3'). The standard curve is a ^{GAPDH amplicon sequence, a derivative of pEGFP-C1 carrying 2.5x10 3} to 2.5x10 ⁷ copies of purified plasmid pP5, an ICAM-1 primer set and an amplicon generated with the plasmid pCR-GAPDH. It was obtained after amplification of the derivative of pCR 2.1 with

The results of these experiments are summarized in Table 2 below.

¹⁾ ON, oligonucleotides.

²⁾ Negative values are associated with sub-uninduced ICAM-1 inhibition in ECV304 cells. These most potent LNA-modified molecules inhibit ICAM-1 expression below basal levels, which is undesirable as summarized above. Only the induced state above the basal level, ie, increased levels of ICAM-1 expression, is considered to be associated with chronic inflammation.

³⁾ gene expression at the level of RNA is comparable to that of protein (Kretschmer-Kazemi Far, R., Sczakiel, G., Nucl. Acids Res., Vol. 31, pp. 4417-4424, (2003) ).

nd, not measurable

---, no measurable target inhibition

In addition, the following Table 3 summarizes the data for the oligonucleotides of SEQ ID NOs: 1-9 provided in Table 2 and provides additional properties of the corresponding oligonucleotides, e.g., with respect to toxicity and immunostimulatory potential as well as cost of production. do.

¹⁾ ON, oligonucleotides.

²⁾ ++, IC ₅₀ < 5 nM; +, IC ₅₀ < 20 nM; -, IC ₅₀ > 20 nM.

³⁾ 2'-OMe is a chemical RNA modification that occurs naturally in RNA. Phosphorothioate chemistries may be toxic at high concentrations when delivered intravenously, but may not, for example, be topically.

⁴⁾ No immune stimulation, as all cytosines are modified (5mC = 2'-deoxy-5-methyl-cytidine).

⁵⁾ The immuno-stimulatory efficacy of the 2'-fluorinated CpG motif remains unclear.

⁶⁾ LNA modification with phosphorothioate backbone showed cell-dependent toxicity.

^{7) The} 2'-F modification is considered toxic. Metabolism in vivo can produce, for example, HF.

All oligonucleotides were characterized for purity and quantity by polyacrylamide gel-electrophoresis and UV absorption measurements. All oligonucleotides were tested for their efficacy in inhibiting ICAM-1 expression in human ECV-304 cells, as described above.

Inhibition of the IL-1β-induced state of ICAM-1 gene expression by asON was measured at the level of ICAM-1 protein and target mRNA. However, both read types behave consistently (Kretschmer-Kazemi-Far, R. et al., Oligonucleotides, Vol. 15, pp. 223-233 (2005); Kretschmer-Kazemi Far, R.,. Sczakiel, G., Nucl. Acids Res., Vol. 31, pp. 4417-4424, (2003)). When IL-1β-stimulated levels are set at 100%, there are basal levels of gene expression in the range of 30% to 40% at the protein level and 15% to 20% at the mRNA level, respectively, depending on the individual experiment.

The important inhibitory properties are summarized in Table 2. Contrary to the general knowledge in the prior art regarding the first and second generation asONs, the antisense oligonucleotide of SEQ ID NO: 1 according to the present invention was found to show high efficacy. Among the settings of the parameters, this is best reflected by the _{low IC 50 values.} In particular, the chemically modified asON targeting the same local target site has _{an IC 50} value of the control of SEQ ID NO: 8 (41 nM _{) compared to the antisense oligonucleotide of SEQ ID NO: 1 (IC 50 : 4 nM) according to the present invention.} ) was a non-strong first order magnitude reflected by (Patzel, V. et al. Patzel, V., Nucl. Acids Res., Vol. 27, pp. 4328-4334 (1999)) (Table 2).

Table 3 provides additional properties of the oligonucleotides of SEQ ID NOs: 1-9. Corresponding data show that, for a local target site of ICAM-1 mRNA directed to CMas1840 (SEQ ID NO: 1), the antisense oligonucleotide of SEQ ID NO: 1 according to the present invention has efficacy, simplicity, toxic and immunostimulatory efficacy, and cost. It proves that it is the best oligonucleotide in terms of

Moreover, the data show that the antisense oligonucleotide of SEQ ID NO: 1 according to the present invention by a miss-match control of SEQ ID NO: 10 and scrambled control of SEQ ID NO: 12 was obtained. It appears to operate according to the predicted "antisense mechanism", supported by the lack of target inhibition (see Table 2).

As evidenced by the above results, the chemical modification of the antisense oligonucleotide of SEQ ID NO: 1 according to the present invention is not known for a major improvement in efficacy, or is even known in the prior art for a lower efficacy (Phosphorus). porothioate) and the chemical modification of the second generation asON, the antisense oligonucleotide of SEQ ID NO: 1 according to the present invention is the second generation and third generation in terms of efficacy, simplicity, toxicity and immunostimulatory efficacy, and cost. surpasses generational chemistry.

SEQUENCE LISTING <110> Universitaet zu Luebeck <120> Antisense drug against the human intercellular adhesion molecule 1 (ICAM-1) <130> IP20214024DE <150> EP 19 153 666.3 <151> 2019-01-25 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapmer oliguncleotide with a central single stranded DNA oligonucleotide sequence flanked on both ends by single stranded 2'-O-methyl RNA oligonucleotide sequences and having phosphorothioate-modified internucleotide phosphates <220> <221> misc_feature <222> (1)..(4) <223> 2'-O-methyladenosine, um, 2'-O-methyl-5-methylcytidine, 2'-O-methyladenosine <220> <221> misc_feature <222> (1)..(20) <223> phosphorothioate-modified internucleotide phosphates <220> <221> misc_feature <222> (9)..(9) <223> 2'-deoxy-5-methylcytidine <220> <221> misc_feature <222> (14)..(15) <223> 2'-deoxy-5-methylcytidine, 2'-deoxy-5-methylcytidine <220> <221> misc_feature <222> (17)..(20) <223> 2'-O-methyladenosine, gm, um, gm <400> 1 ancagatgcg tggcctannn 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> central oligonucleotide sequence with monophosphorothioate internucleotide linkages flanked on both ends by locked nucleic acids <220> <221> misc_feature <222> (1)..(4) <223> locked nucleic acid modified nucleotides <220> <221> misc_feature <222> (4)..(16) <223> phosphorothioate-modified internucleotide phosphates between nucleotides 4 to 16 <220> <221> misc_feature <222> (17)..(20) <223> locked nucleic acid modified nucleotides <400> 2 atcagatgcg tggcctagtg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide with 2'-fluoro-modified riboses <220> <221> misc_feature <222> (1)..(20) <223> 2'-fluoro-modified riboses <400> 3 atcagatgcg tggcctagtg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapmer oliguncleotide with a central single stranded oligonucleotide sequence flanked on both ends by single stranded 2'-fluoro-modified oligonucleotide sequences and having phosphorothioate-modified internucleotide phosphates <220> <221> misc_feature <222> (1)..(4) <223> 2'-fluoro-modified riboses <220> <221> misc_feature <222> (1)..(20) <223> phosphorothioate-modified internucleotide phosphates <220> <221> misc_feature <222> (17)..(20) <223> 2'-fluoro-modified riboses <400> 4 atcagatgcg tggcctagtg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide with 2'-O-methyl-modified riboses <220> <221> misc_feature <222> (1)..(20) <223> 2'-O-methyl-modified riboses <400> 5 atcagatgcg tggcctagtg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide having 2'-O-methyl-modified riboses and having phosphorothioate-modified internucleotide phosphates <220> <221> misc_feature <222> (1)..(20) <223> 2'-O-methyl-modified riboses <220> <221> misc_feature <222> (1)..(20) <223> phosphorothioate-modified internucleotide phosphates <400> 6 atcagatgcg tggcctagtg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapmer oliguncleotide with a central single stranded oligonucleotide sequence flanked on both ends by single stranded 2'-O-methyl-modified oligonucleotide sequences and having phosphorothioate-modified internucleotide phosphates <220> <221> misc_feature <222> (1)..(20) <223> phosphorothioate-modified internucleotide phosphates <220> <221> misc_feature <222> (1)..(4) <223> 2'-O-methyl-modified riboses <220> <221> misc_feature <222> (17)..(20) <223> 2'-O-methyl-modified riboses <400> 7 atcagatgcg tggcctagtg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide having phosphorothioate-modified internucleotide phosphates <220> <221> misc_feature <222> (1)..(20) <223> phosphorothioate-modified internucleotide phosphates <400> 8 atcagatgcg tggcctagtg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapmer oliguncleotide with a central single stranded DNA oligonucleotide sequence flanked on both ends by single stranded 2'-O-methyl RNA oligonucleotide sequences and having phosphorothioate-modified internucleotide phosphates <220> <221> misc_feature <222> (1)..(4) <223> 2'-O-methyladenosine, um, cm, 2'-O-methyladenosine <220> <221> misc_feature <222> (1)..(20) <223> phosphorothioate-modified internucleotide phosphates <220> <221> misc_feature <222> (17)..(20) <223> 2'-O-methyladenosine, gm, um, gm <400> 9 annagatgcg tggcctannn 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapmer oliguncleotide with a central single stranded oligonucleotide sequence flanked on both ends by single stranded 2'-O-methyl-modified oligonucleotide sequences and having phosphorothioate-modified internucleotide phosphates <220> <221> misc_feature <222> (1)..(4) <223> 2'-O-methyladenosine, tm, cm, 2'-O-methyladenosine <220> <221> misc_feature <222> (1)..(20) <223> phosphorothioate-modified internucleotide phosphates <220> <221> misc_feature <222> (17)..(20) <223> 2'-O-methyladenosine, gm, tm, gm <400> 10 annagattcg gggcctannn 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> unmodified DNA <400> 11 atcagatgcg tggcctagtg 20 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide having phosphorothioate-modified internucleotide phosphates <220> <221> misc_feature <222> (1)..(18) <223> phosphorothioate-modified internucleotide phosphates <400> 12 ggtcagacca gtgagttc 18 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for ICAM-1 cDNA <400> 13 gccacttctt ctgtaagtct gtggg 25 <210> 14 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for ICAM-1 cDNA <400> 14 ctaccggccc tgggacg 17 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for cDNA encoding human GAPDH <400> 15 aacagcgaca cccactcctc 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for cDNA encoding human GAPDH <400> 16 ggaggggaga ttcagtgtgg t 21

Claims (15)

An antisense oligonucleotide comprising the nucleotide sequence according to SEQ ID NO: 1 or a fragment thereof. The antisense oligonucleotide of claim 1 , for use in a method of preventing or treating a disease or condition in a human, wherein the disease or condition is associated with overexpression of ICAM-1. 3. The method of claim 2, wherein the disease or condition is an acute or chronic inflammatory disease or condition, viral infection, metastasis, inflammation of the skin, mobilization of hematopoietic stem cells, rhinitis, ulcerative colitis, rheumatoid arthritis, lupus erythematosus, organ rejection reaction, graft-versus-host reaction to allogeneic bone marrow transplantation, psoriasis, asthma, and neurodermatitis. The antisense oligonucleotide according to any one of claims 1 to 3, wherein the oligonucleotide has a length of 10 to 50 bases. The antisense oligonucleotide according to any one of claims 1 to 3, wherein the antisense oligonucleotide consists of a nucleotide sequence according to SEQ ID NO: 1. 6. The antisense oligonucleotide according to any one of claims 1 to 5, wherein the antisense oligonucleotide is directed against a specific nucleic acid sequence of ICAM-1. 7. The down-regulate according to claim 6, wherein the stimulated range of ICAM-1 gene expression is 40% or less and -10% or more, based on 100% stimulated ICAM-1 gene expression by the antisense oligonucleotide. ), which is an antisense oligonucleotide. A vector comprising an antisense oligonucleotide according to any one of claims 1 to 7. A host cell comprising the vector according to claim 8 or the antisense oligonucleotide according to any one of claims 1 to 7. A pharmaceutical composition comprising a pharmaceutically acceptable amount of an antisense oligonucleotide according to any one of claims 1 to 7, and optionally a pharmaceutically acceptable carrier, excipient or diluent. A kit comprising the antisense oligonucleotide according to any one of claims 1 to 7. Use of an antisense oligonucleotide according to any one of claims 1 to 7 for reducing the expression of ICAM-1. A method for reducing the expression of ICAM-1, comprising introducing the antisense oligonucleotide according to any one of claims 1 to 7 or the vector according to claim 8 into a human cell. The use according to claim 12 or the method according to claim 13 , wherein the use or method is carried out in vitro. 15. Use according to claim 12 or 14 or according to claim 13 or 14, wherein the expression of ICAM-1 is reduced by 40% or less and -10% or more, based on 100% stimulated ICAM-1 gene expression. how to follow.

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